Three predominant isoenzymes of creatine kinase (CK; E.C. 2.7.3.2) are recognized; these are dimers consisting of the M and B sub-units. These dimers may comprise two M or two B sub-units, or one M and one B sub-unit. The predominant dimer present in the blood, serum, or plasma of normal individuals is CK-MM isoenzyme, with variable but usually only trace quantities of CK-MB that reflect the normal degradation of skeletal muscle. The CK-BB isoenzyme is not usually present in detectable amounts in serum of normal persons but is present in significant quantities in brain tissue and smooth muscle. Elevations of the BB isoenzyme can occur in pathologic conditions such as metastatic carcinoma or severe burns. The presence of elevated levels of CK-MB isoenzyme has been used as a clinically important indication of myocardial infarction in instances where possible sources of significant skeletal muscle damage can be eliminated. More particularly, repetitive determinations of CK-MB level in the serum can indicate the time course and severity of infarctions. Differentiation between the isoenzymes of creatine kinase, therefore, is clinically important and the availability of a rapid, efficient, and highly discriminatory assay for the CK isoenzymes was needed.
Several approaches to analysis of the CK isoenzymes have been used in the past which rely either upon physical separation of the isoenzymes with subsequent identification, or upon highly selective reactions between the isoenzymes and antibodies. Physical separation methods such as electrophoresis or column chromatography are time consuming, require considerable skill, and are frequently incapable of highly reproducible separations to resolve adequately the isoenzymes with sufficient sensitivity to monitor early changes in CK-MB levels. The inconvenience of physical separation techniques and their inability to unequivocally resolve CK isoenzymes lead to immunochemical techniques which, based upon their unique structural or immunochemical determinants, have the potential to differentiate between the isoenzymes in complex mixtures.
Jockers-Wretou et al., Clin. Chem. Acta Volume 58,223 (1975), used an immunoprecipitation method with antisera elicited in rabbits by the crystallized MM and BB isoenzymes from human muscle and brain tissue, respectively. Both forms of isoenzyme were quantitatively precipitated from tissue extracts and serum without measurable cross reaction. The CK-MB isoenzyme reacted to some degree with both antisera, but not completely with either. It was also demonstrated that the CK-MM isoenzyme activity was completely inhibited after reaction with anti-CK-MM antibody. The CK-MB isoenzyme activity was inhibited by 80% when reacted with either anti-CK-BB or anti-CK-MM antiserum. The CK-BB isoenzyme was not completely inhibited by binding to anti-CK-BB antibody, but could be completely removed by centrifugation. Therefore, the combination of selective immunoprecipitation and immunoinhibition of the isoenzymes allowed their differentiation, but the process required separate centrifugations and more than one activity assay to determine the relative contributions of the isoenzymes, particularly the CK-MB activity, by comparison to CK-MM and CK-BB samples. In addition, about 10% of the CK-MB activity was neither precipitable nor inhibitable by the disclosed procedures, which introduced error into enzyme sub-unit estimates.
U.S. Pat. No. 4,067,775, issued Jan. 10, 1978 to Wurzburg et al., discloses an immunoinhibition assay for CK-MB in which antibodies capable of completely inhibiting the M sub-unit activity were combined with an appropriate sample in solution. The mixture was allowed to react for sufficient time to substantially or completely inhibit the M sub-unit and the residual enzyme activity in the solution was determined. This method represented an advancement by use of a nonprecipitating antibody which obviated the need for long incubations and centrifugations, and also made possible direct testing of the uninhibited B sub-unit in solution without additional sample processing. This approach offers speed and simplicity, but is subject to the disadvantage that, because the B sub-unit is essentially uninhibited, any contaminating BB isoenzyme in the sample can provide a source of error in those instances where concentrations are significant. A typical CK isoenzymes, such as mitochondrial or macro CK types 1 and 2, can also provide sources of error. The activity contributed by adenylate kinase must be determined in a separate reaction medium which lacks phosphocreatine, which is the normal CK substrate, and this activity value has to be subtracted from the antibody-treated reaction mixture activity value. These various elements introduce sources of error in the determination of CK-MB activity and require additional control assays to blank contaminating adenylate kinase activities.
U.S. Pat. No. 4,260,678, issued Apr. 7, 1981 to Lepp et al., discloses a CK assay using immobilized antibodies to either the CK-M or CK-B sub-unit, or both. The selected antibody, which did not inhibit or substantially change the activity of the bound sub-unit, was immobilized on a carrier such as porous glass beads and then reacted with sample followed by the separation of the immobilized antibody-isoenzyme complexes from the reaction mixture prior to determination of enzyme activity of sub-units bound to the carrier. This approach is useful to determine total activity by reacting both anti-CK-M and anti-CK-B carriers with sample, or of individual sub-units by reaction of one or the other carrier with sample. This method allows separation of desired isoenzymes from contaminating forms, such as macro-CK, but also requires multiple assays with subtraction of results to determine activity associated with each sub-unit type. It is not possible to determine the activity associated with the hybrid dimer CK-MB using this approach. Therefore, the technique has limited utility in a clinical laboratory.
U.S. Pat. No. 4,387,160, issued June 3, 1983 to Gomez et al., discloses an assay for CK-MB which uses three separate antibody preparations and two separate assays on a given sample. In one assay, an anti-CK-M antibody capable of substantially or completely inhibiting the M sub-unit activity, without significantly affecting B sub-unit activity, is combined with one portion of a sample and allowed to react. If precipitation occurs during this reaction, the precipitate will remain homogeneously suspended during the process of this reaction. The residual isoenzyme activity in this solution is determined by conventional means. A second portion of sample containing at least CK-M sub-units is reacted with an anti-CK-M antibody and the complexes so formed are further reacted with a precipitating second antibody capable of reacting with determinants on the anti-CK-M antibody. The precipitate is separated from the reaction mixture and residual activity, presumably representing B sub-unit remaining in the supernatant, is determined by conventional means. Activity from the second sample representing contaminating B sub-units is subtracted from the activity of the first sample, which represents both MB and contaminating B sub-units, to determine MB activity. This assay combines immunoinhibition with precipitation techniques to determine CK-MB levels in samples, but does so with considerable inconvenience in time-consuming processing steps and with several, highly specialized reagents.
Finally, sandwich immunoassays employing the two-site immunometric approach to measure mass concentrations, not enzymatic activity, see U.S. Pat. No. 4,376,110, issued Mar. 8, 1983, can be used to detect CK-MB by immobilizing all M or B-containing sub-units onto a solid phase by binding to a monoclonal antibody specific for one sub-unit that is itself covalently attached to the solid phase. The desired sub-unit is detected by binding with a labelled second monoclonal antibody specific for that sub-unit. The amount of label, such as enzyme activity, is a direct measure of the concentration of the desired sub-unit in the original sample. This approach requires two highly specific reagents, one labelled with an appropriate indicator enzyme, and can be subject to an elevated number of false positive results owing to the interaction of the enzyme-labelled second antibody with the solid support in a nonspecific manner. This disadvantage can be overcome by the analysis of inherent CK sub-unit acitivity to determine levels of CK-MB under appropriate circumstances.
There is a need for a CK-MB assay that can provide simply, precisely, and economically an accurate evaluation of the CK-MB hybrid dimer level in body fluids of individuals suspected of suffering from acute myocardial infarction.